1. Controlling Procaryotic Transcription Lecture 12

2. Differential Gene Expression Some proteins present at 100,000 copies per cell –Others only at <10 copies per cell Consequence of strong and weak promoters –Close to consensus sequence tend to be transcribed more frequently Every 2 seconds –Poorer fits for sigma give weaker promoters Changes in the TTGACA or the TATAAT or the spacing between them Only initiated once every 10 mins Constitutive vs Inducible/Repressible –Expressed at a constant number per cell at all times “Housekeeping genes” –Others are turned on/off at different times

3. Contributing factors Cis factors only affect one gene –local –Eg, DNA sequences around the promoter Trans factors affect many genes –global –proteins in addition to RNA pol DNA binding activators – recruit RNA pol Repressors which prevent RNA pol from binding Also called TRANSCRIPTION FACTORS

4. Activators Activating accessory proteins can bind near the promoter –Eg, CAP (catabolite activator protein) binds at -80 of several genes –Interacts with alpha subunit of RNA pol and brings RNA pol into the promoter In bugs these sequences are called Proximal Promoter Elements –In eucaryotes, the regulatory sequences can be miles away! Called enhancer sequences Remember the hugely coiled nature of eucaryotic DNA

5. Repressors Repressing proteins can also bind near the promoter –Often close to the start (eg, -10) –So they block the RNA pol Balance between activators and repressors determines the rate of transcription

6. Modulating the Factors The binding affinity of the activators and repressors for DNA can change –As can their ability to activate or block RNA pol –Most often change is through binding smaller molecules Classic examples –The lac system codes for the genes necessary for lactose breakdown Normally bugs don’t encounter lactose but, if it’s around, it makes sense to make the necessary enzymes! –The trp system codes for the genes necessary for tryptophan synthesis Normally bugs need to make this amino acid but, if it’s around, it makes sense not to bother making all the enzymes

7. Keeping lac genes repressed A repressor protein is continually (constitutively) made The repressor binds to a sequence in front of the normal promoter and blocks RNA pol Normally bugs do not make the enzymes necessary for lactose metabolism repressor Genes for lactose metabolismPromoter region

8. Lactose Derepression RNA pol is free to transcribe the gene. repressor Genes for lactose metabolismPromoter region The repressor has a binding site for lactose. When lactose is bound, the repressor no longer binds to the DNA. RNA pol Lactose arrives

9. Activating lac genes But the promoter is weak (quite different from the consensus sequence). RNA pol needs a hand to get going. The binding of CAP upstream is needed. But CAP won’t bind if the cell is already using a better fuel (eg, glucose). How does CAP know whether it should be binding or not? Genes for lactose metabolismPromoter region RNA pol CAP

10. Expression of lac genes Genes for lactose metabolismPromoter region RNA pol CAP cAMP When cAMP is bound to CAP, RNA pol is able to transcribe the lac genes cAMP is low when the cells are using glucose but is high when the cells are using inferior fuels. The interaction is between the CAP and the alpha subunit of RNA pol In order to bind to the promoter, CAP needs to binds a molecule called cyclic AMP (cAMP)

11. Control of Biosynthetic Genes The repressor is produced continually (constitutively) at low level The repressor doesn’t bind to the promoter, so RNA pol is free to do it’s job The default position is that bugs want to make the enzymes necessary for creating the amino acids repressor Genes for amino acid productionPromoter region RNA pol The promoter is nice and strong… so transcription occurs easily.

12. Control of Biosynthetic Genes The repressor now binds to the DNA and blocks RNA pol. When there is plenty of the amino acid around, there’s no point in transcribing the genes for its synthesis. Amino acid (trp) Genes for amino acid productionPromoter region When the amino acids are around, they bind to the repressor

13. General Themes Genes coding for degradative enzymes –Normally repressed unless they are needed Genes coding for biosynthetic enzymes –Normally expressed unless there’s plenty of the product around Note how the affinity of the protein for DNA changes several thousand fold by binding the compound A protein exerts POSITIVE control –When its binding causes an event –Eg, activator like CAP A protein exerts NEGATIVE control –When its binding prevents an event –Eg, repressor A gene is never totally off (except in spores!)

14. Changing Large Groups of Genes One type of protein can affect many genes –CAP has a binding site on many genes –So several genes are switched on when it is activated For polycistronic mRNAs, many genes affected by one promoter –Groupings of several genes behind one promoter are called OPERONS Trp operon and lac operon Sometimes whole panels of genes need to be switched on –Nitrogen starvation genes have CTGGNAN 6 TTGCA promoters When faced with N-starvation a special sigma (σ 60 ) is produced that recognises the specific promoters –Heat shock genes have CNCTTGAAN 14 CCCATNT promoters Recognised by σ 32 which, itself, if produced when the temperature changes –Bacteriophages (viruses which invade bugs) produce their own sigma proteins Allow their own genes to be expressed in preference to their hosts

15. Mutations in Control Processes Classic exam question What happens when there’s a mutation? –In the promoter sequence? –In the proximal promoter or repressor binding sites? –In the activator or repressor proteins? –In the gene products?

16. Text Book Refs Chapter 11 –p223 – opening paragraphs on why gene regulation is so exciting –p224 - principles explained pretty well –p225 – positive and negative regulation explained –DO NOT FRET OVER p226-229 on the lac operon – it is too detailed –But on p230, the numbered points are manageable –Fig 11-6 is OK –p232 – the stuff on CAP (called CRP in your book) is too detailed.. but read it anyway –p236 on the trp operon is a bit hard but the first paragraph on p237 gives the principles pretty well –p240 – is good on the differences between global and specific –p241 – the section on constitutive, housekeeping genes –NB: I don’t think there’s any references in the book to the special sigma subunits used to regulate heat-shock, N-starvation or bacteriophage expression.